Objective-The goal of this study was to assess the role of B-cell activating factor (BAFF) receptor in B-cell regulation of atherosclerosis. Methods and Results-Male LDL receptor-deficient mice (Ldlr −/− ) were lethally irradiated and reconstituted with either wild type or BAFF receptor (BAFF-R)-deficient bone marrow. After 4 weeks of recovery, mice were put on a high-fat diet for 6 or 8 weeks. BAFF-R deficiency in bone marrow cells led to a marked reduction of conventional mature B2 cells but did not affect the B1a cell subtype. This was associated with a significant reduction of dendritic cell activation and Tcell proliferation along with a reduction of IgG antibodies against malondialdehyde-modified low-density lipoprotein. In contrast, serum IgM type antibodies were preserved. Interestingly, BAFF-R deficiency was associated with a significant reduction in atherosclerotic lesion development and reduced numbers of plaque T cells. Selective BAFF-R deficiency on B cells led to a similar reduction in lesion size and T-cell infiltration but in contrast did not affect dendritic cell activation. Conclusion-BAFF-

Altogether, our data demonstrate that regulation of proangiogenic Ly6C(hi) monocytes systemic levels by CCL2/CCR2 controls post-ischaemic vessel growth, whereas Ly6C(lo) monocytes have no major role in this setting.

Background-CD4ϩ and CD8 ϩ T lymphocytes are key regulators of postischemic neovascularization. T-cell activation is promoted by 2 major costimulatory signalings, the B7/CD28 and CD40 -CD40 ligand pathways. Interestingly, CD28 interactions with the structurally related ligands B7-1 and B7-2 are also required for the generation and homeostasis of CD4 ϩ CD25 ϩ regulatory T cells (Treg cells), which play a critical role in the suppression of immune responses and the control of T-cell homeostasis. We hypothesized that Treg cell activation may modulate the immunoinflammatory response to ischemic injury, leading to alteration of postischemic vessel growth. Methods and Results-Ischemia was induced by right femoral artery ligation in CD28-, B7-1/2-, or CD40-deficient mice (nϭ10 per group). CD40 deficiency led to a significant reduction in the postischemic inflammatory response and vessel growth. In contrast, at day 21 after ischemia, angiographic score, foot perfusion, and capillary density were increased by 2.0-, 1.2-, and 1.8-fold, respectively, in CD28-deficient mice, which showed a profound reduction in the number of Treg cells compared with controls. Similarly, disruption of B7-1/2 signaling or anti-CD25 treatment and subsequent Treg deletion significantly enhanced postischemic neovascularization. These effects were associated with enhanced accumulation of CD3-positive T cells and Mac-3-positive macrophages in the ischemic leg. Conversely, treatment of CD28 Ϫ/Ϫ mice with the nonmitogenic anti-CD3 monoclonal antibody enhanced the number of endogenous Treg cells and led to a significant reduction of the postischemic inflammatory response and neovascularization. Finally, coadministration of Treg cells and CD28 Ϫ/Ϫ splenocytes in Rag1 Ϫ/Ϫ mice with hindlimb ischemia abrogated the CD28 Ϫ/Ϫ splenocyte-induced activation of the inflammatory response and neovascularization.

Background-The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O 2 -dependent prolyl hydroxylase domain proteins (PHD1, PHD2, and PHD3). We investigated whether inhibition of PHDs via upregulating HIF might promote postischemic neovascularization. Methods and Results-Mice with right femoral artery ligation were treated, by in vivo electrotransfer, with plasmids encoding for an irrelevant short hairpin RNA (shRNA) (shCON [control]) or specific shRNAs directed against HIF-1␣ (shHIF-1␣), PHD1 (shPHD1), PHD2 (shPHD2), and PHD3 (shPHD3). The silencing of PHDs induced a specific and transient downregulation of their respective mRNA and protein levels at day 2 after ischemia and, as expected, upregulated HIF-1␣. As a consequence, 2 key hypoxia-inducible proangiogenic actors, vascular endothelial growth factor-A and endothelial nitric oxide synthase, were upregulated at the mRNA and protein levels. In addition, monocyte chemotactic protein-1 mRNA levels and infiltration of Mac-3-positive macrophages were enhanced in ischemic leg of mice treated with shPHD2 and shPHD3. Furthermore, activation of HIF-1␣-related pathways was associated with changes in postischemic neovascularization. At day 14, silencing of PHD2 and PHD3 increased vessel density by 2.2-and 2.6-fold, capillary density by 1.8-and 2.1-fold, and foot perfusion by 1.2-and 1.4-fold, respectively, compared with shCON (PϽ0.001). shPHD1 displayed a lower proangiogenic effect. Of interest, coadministration of shHIF-1␣ with shPHD3 abrogated shPHD3-related effects, suggesting that activation of endogenous HIF-1-dependent pathways mediated the proangiogenic effects of PHD silencing. Conclusions-We demonstrated that a direct inhibition of PHDs, and more particularly PHD3, promoted therapeutic revascularization. Furthermore, we showed that activation of the HIF-1 signaling pathway is required to promote this revascularization. (Circulation. 2009;120:50-59.)

Key Words: hypertension Ⅲ angiogenesis Ⅲ progenitor cells Ⅲ angiotensin converting enzyme Ⅲ angiotensin type I receptor T he revascularization process, including vasculogenesis, angiogenesis, and collateral growth, characterizes tissue repair and remodeling occurring in acute and chronic ischemic vascular diseases. In particular, postnatal vasculogenesis referred to the homing and differentiation of circulating progenitor cells from bone marrow or non-bone marrow origins 1 into endothelial cells within sites of active neovascularization. In addition, circulating progenitor cells may deliver angiogenic growth factors to pathological tissues and contribute to neovascularization and tissue/vessel remodeling by paracrine effects. 2,3 In most clinical settings, however, these natural adaptive responses to a compromised perfusion are insufficient to block the progression of ischemic diseases. Hence, certain cardiovascular risk factors including diabetes, aging, and hypercholesterolemia adversely affect postnatal vasculogenesis and revascularization in animals models of limb ischemia. 4 -7 In support of this view, patients with type I and II diabetes displayed a reduction in endothelial progenitor cell (EPC) number and angiogenicity. 8,9 In most forms of clinical and experimental hypertension, increased arterial blood pressure is associated with microvascular rarefaction and increased peripheral vascular resistances. 10 Similarly, postischemic reparative neovascularization is impaired in spontaneously hypertensive rats (SHR) as a function of progression of the hypertensive disease. 11,12 Several molecular and cellular mechanisms may be involved in the hypertension-induced impairment in vessel growth. First, the angiogenic capacity of serum derived from SHR was less than that from normotensive animals in a chick embryo chorio-allantoic membrane model. 13 Protein levels of key proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor are also reduced in hypertensive animals. 11,14 Second, previous obser-

Bone marrow-derived mononuclear cells (BMMNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. However, cardiovascular risk factors, including diabetes mellitus and hypercholesterolemia, lead to the abrogation of BMMNCs proangiogenic potential. NO has been shown to be critical for the proangiogenic function of BMMNCs, and increased endothelial NO synthase (eNOS) activity promotes vessel growth in ischemic conditions. We therefore hypothesized that eNOS overexpression could restore both the impaired neovascularization response and decreased proangiogenic function of BMMNCs in clinically relevant models of diabetes and hypercholesterolemia. To prevent or treat ischemic diseases, therapeutic neovascularization, the stimulation of tissue vascularization after ischemia, has recently progressed from the bench to the bedside. Strategies include transplantation of angiogenic bone marrow-derived mononuclear cells (BMMNCs) or gene transfer for systemic or local up-regulation of proangiogenic proteins. Clinical studies have demonstrated the safety, feasibility, and efficacy of intracoronary and intramuscular infusion of adult BMMNCs in patients with peripheral arterial disease, acute myocardial infarction, and ischemic cardiomyopathy. 1,2However, despite the excitement surrounding the possible clinical use of BMMNCs, in atherosclerosis, diabetes mellitus, and other risk factors for cardiovascular diseases the availability of bone marrow and progenitor cells is reduced and their function impaired to varying degrees.1,2 Moreover, the safety of BMMNCs treatment has been questioned by studies that found an increase in atherosclerotic plaque size after BMMNCs treatment. 3This potentially hazardous dual effect of therapeutic neovascularization on atherogenesis is explained by the many common pathways of both mechanisms and has been named the Janus phenomenon.

Upregulation of hypoxia-inducible transcription factor-1a (HIF-1a), through prolyl-hydroxylase domain protein (PHD) inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodeling. We aimed to unravel the effect of specific PHD2 isoform silencing in cell-based strategies designed to promote therapeutic revascularization in patients with critical limb ischemia (CLI). PHD2 mRNA levels were upregulated whereas that of HIF-1a were downregulated in blood cells from patients with CLI. We therefore assessed the putative beneficial effects of PHD2 silencing on human bone marrow-derived mesenchymal stem cells (hBM-MSC)-based therapy. PHD2 silencing enhanced hBM-MSC therapeutic effect in an experimental model of CLI in Nude mice, through an upregulation of HIF-1a and its target gene, VEGF-A. In addition, PHD2-transfected hBM-MSC displayed higher protection against apoptosis in vitro and increased rate of survival in the ischemic tissue, as assessed by Fluorescence Molecular Tomography. Cotransfection with HIF-1a or VEGF-A short interfering RNAs fully abrogated the beneficial effect of PHD2 silencing on the proangiogenic capacity of hBM-MSC. We finally investigated the effect of PHD2 inhibition on the revascularization potential of ischemic targeted tissues in the diabetic pathological context. Inhibition of PHD-2 with shRNAs increased postischemic neovascularization in diabetic mice with CLI. This increase was associated with an upregulation of proangiogenic and proarteriogenic factors and was blunted by concomitant silencing of HIF-1a. In conclusion, silencing of PHD2, by the transient upregulation of HIF-1a and its target gene VEGF-A, might improve the efficiency of hBM-MSC-based therapies. STEM CELLS 2014;32:231-243

Background— C/EBP homologous protein-10 (CHOP-10) is a novel developmentally regulated nuclear protein that emerges as a critical transcriptional integrator among pathways regulating differentiation, proliferation, and survival. In the present study, we analyzed the role of CHOP-10 in postnatal neovascularization. Methods and Results— Ischemia was induced by right femoral artery ligation in wild-type and CHOP-10 −/− mice. In capillary structure of skeletal muscle, CHOP-10 mRNA and protein levels were upregulated by ischemia and diabetes mellitus. Angiographic score, capillary density, and foot perfusion were increased in CHOP-10 −/− mice compared with wild-type mice. This effect was associated with a reduction in apoptosis and an upregulation of endothelial nitric oxide synthase (eNOS) levels in ischemic legs of CHOP-10 −/− mice compared with wild-type mice. In agreement with these results, eNOS mRNA and protein levels were significantly upregulated in CHOP-10 short interfering RNA–transfected human endothelial cells, whereas overexpression of CHOP-10 inhibited basal transcriptional activation of the eNOS promoter. Using a chromatin immunoprecipitation assay, we also showed that CHOP-10 was bound to the eNOS promoter. Interestingly, enhanced postischemic neovascularization in CHOP-10 −/− mice was fully blunted in CHOP-10/eNOS double-knockout animals. Finally, we showed that induction of diabetes mellitus is associated with a marked upregulation of CHOP-10 that substantially inhibited postischemic neovascularization. Conclusions— This study identifies CHOP-10 as an important transcription factor modulating vessel formation and maturation.